Retro-inverso peptides derived from interleukin-3

ABSTRACT

The invention provides methods of treatment using retro-inverso peptides derived from interleukin-3 (IL-3) having between 12 and about 40 amino acids and including the sequence that is retro-inverso with respect to SEQ ID NO: 1. The peptides of the invention have the same activity as native IL-3 and also have neurotrophic activity. The peptides of the invention are also less susceptible to proteolytic degradation in vivo because of their D-amino acid linkage.

This application is a continuation of U.S. patent application Ser. No.10/048,302, filed May 7, 2002, which is a national phase application ofInternational Patent Application No. PCT/US00/16759, filed Jun. 16,2000, which claims priority to U.S. Provisional Patent Application No.60/139,688 filed on Jun. 16, 1999, each of which is expresslyincorporated herein in its entirety by reference thereto.

FIELD OF THE INVENTION

The present invention relates to retro-inverso peptides derived frominterleukin-3 (IL-3). These peptides have activities similar to that ofthe native parent protein, and also have neurotrophic activity.

BACKGROUND OF THE INVENTION

Cytokines are proteins which are produced during the effector phases ofnatural and specific immunity and serve to mediate and regulate immuneand inflammatory responses. Cytokines, like other polypeptide hormones,initiate their action by binding to specific receptors on the surface oftarget cells. One of the most well known families of cytokines are theinterleukins which mediate natural immunity. For a detailed descriptionof the structure and function of the interleukins, see Abbas et al.,Cellular and Molecular Immunology, W. B. Saunders Company, Philadelphia,pp. 225-243 (1991).

IL-3 acts on numerous target cells within the hematopoietic system. Adetailed review of the structure and function of this cytokine may befound in The Cytokine Handbook, Third Edition, Thomson, A. Ed., AcademicPress, San Diego, Calif. (1998). IL-3 is a glycoprotein having broadstructural similarities with other interleukins and hematopoietic growthfactors. Murine IL-3 contains 140 amino acids, while human IL-3 contains133 amino acids. The amino acid sequences of mouse and humanIL-3 exhibitonly 30% identity, reflecting the lack of cross-species biologicactivity (Yang et al., Cell, 47: 3-10 (1986)).

IL-3 has the broadest target specificity of any of the hematopoieticgrowth factors. The range of target cells includes progenitor cells ofevery lineage derived from the pluripotential hematopoietic stem cells.Thus, IL-3 can stimulate the generation and differentiation ofmacrophages, neutrophils, eosinophils, basophils, mast cells,megakaryocytes and erythroid cells. In vitro, hematopoietic stem andprogenitor cells rapidly die if cultured in tissue culture medium alone.Like other hematopoietic growth factors, IL-3 prevents death byapoptosis and promotes survival in vitro (Williams et al., Nature, 343:76-79 (1990). When deprived of IL-3, IL-3-dependent cells undergoapoptosis (Williams et al., supra.).

The subcutaneous administration of 2000 ED₅₀ units of IL-3 three times aday for three days resulted in an increase in splenic weight and in thenumber of mast cells and the progenitors of mast cells, neutrophils andmacrophages (Schrader et al., Immune Regulation by CharacterizedPolypeptides, Goldstein, G. et al., eds., Liss, New York, pp. 475-484(1986)). The administration of humanIL-3 to primates and humans resultedin similar effects to those seen in mice (Donahue et al., Science, 241:1820-1823 (1988); Mayer et al., Blood, 74: 613-621 (1989)). Incynomolgus monkeys, IL-3 induced extramedullary hematopoiesis at sitesof subcutaneous injection (Khan et al., Toxicol. Pathol., 24: 391-397(1996)). IL-3 may have particular utility in stimulating plateletproduction (Ganser et al., Blood, 76: 666-676 (1990)). In addition,clinical trials suggest that sequential administration of IL 3 and G-CSFor GM-CSF may provide optimal stimulation of myelopoiesis (Lemoli etal., J. Clin. Oncol., 14: 3018-3025 (1996).

Neurotrophins and neurotrophic factors are proteins or peptides capableof affecting the survival, target innervation and/or function ofneuronal cell populations (Barde, Neuron, 2: 1525-1534 (1989)). Theefficacy of neurotrophins both in vivo and in vitro has been welldocumented. For example, ciliary neurotrophic factor (CNTF) promotessurvival of chicken embryo ciliary ganglia in vitro and supportssurvival of cultured sympathetic, sensory and spinal motor neurons (lpet al., J. Physiol. Paris, 85:123-130 (1991)).

A major obstacle to the in vivo therapeutic use of peptides is theirsusceptibility to proteolytic degradation. The half-life ofintravenously injected IL-3 is short, being on the order of only 40minutes (Crapper et al., Immunology, 53: 33-42 (1984)). Retro-inversopeptides are isomers of linear peptides in which the direction of thesequence is reversed (retro) and the chirality, D or L, of each aminoacid is inverted (inverso). There are also partially modifiedretro-inverso isomers of linear peptides in which only some of thepeptide bonds are reversed and the chirality of the amino acid residuesin the reversed portion is inverted. The major advantage of suchpeptides is their enhanced activity in vivo due to improved resistanceto proteolytic degradation (For review, see Chorev et al., TrendsBiotech., 13: 438-445 (1995)). Although such retro-inverso analogsexhibit increased metabolic stability, their biological activity isoften greatly compromised (Guichard et al., Proc. Natl. Acad. Sci.U.S.A., 91: 9765-9769 (1994)). For example, Richman et al. (J. PeptideProtein Res., 25: 648-662) determined that analogs of linear and cyclicleu-enkephalin modified at the Gly³-Phe⁴ amide bond had activitiesranging from 6%-14% of native leu-enkephalin. Chorev et al., (ibid.)showed that retro-inversion of a peptide which inhibits binding ofvitronectin to its receptor resulted in one peptide which was lesspotent than the parent isomer by a factor of 50,000, and another peptidewhich was 4,000 fold more potent than the parent cyclic peptide.Guichard et al. (TIBTECH 14 (1996)), teach that retro-inverso(all-D-retro) antigenic mimicry may only occur with peptides in randomcoil, loop or cyclic conformations. In the case of “helical” peptide,adequate, functional mimicry would be expected only if the helicity was,in fact, absent under the solvent conditions used for assessingantigenic mimicry.

There is a need for IL-3-derived and neurotrophic peptides exhibitingincreased metabolic stability while retaining biological activity. Thepresent invention addresses this need.

SUMMARY OF THE INVENTION

One embodiment of the present invention is an isolated retro-inversopeptide having between 12 and about 40 amino acids, wherein said peptideincludes the sequence that is retro-inverso with respect to SEQ ID NO:1, i.e. wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.In one aspect of this preferred embodiment, at least one basic chargedamino acid of said sequence is replaced with a different basic chargedamino acid. In another aspect of this preferred embodiment, at least oneacidic charged amino acid of said sequence is replaced with a differentacidic charged amino acid. Advantageously, at least one non-polar aminoacid of said sequence is replaced with a different non-polar amino acid.Preferably, at least one uncharged amino acid of said sequence isreplaced with a different uncharged amino acid. In another aspect ofthis preferred embodiment, at least one aromatic amino acid of saidsequence is replaced with a different aromatic amino acid.Advantageously, the peptide is modified at the amino terminus, carboxyterminus, or both amino and carboxy terminus with a moiety independentlyselected from the group consisting of CH₃CO, CH₃(CH₂)_(n)CO, C₆H₅CH₂COand H₂N(CH₂)_(n)CO, wherein n=1-10. Preferably, the peptide isglycosylated. In another aspect of this preferred embodiment, one ormore amide bonds of the peptide are reduced. Preferably, one or morenitrogens in said peptide are methylated. In still another aspect ofthis preferred embodiment, one or more carboxylic acid groups in thepeptide are esterified. Preferably, the peptide has the amino acidsequence that is retro-inverso with respect to SEQ ID NO: 1, i.e.wherein said peptide consists of the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.

The present invention also provides a method for promoting neuriteoutgrowth or myelination in a mammal in need thereof, comprising thestep of administering to the mammal an effective, neurite outgrowth ormyelination facilitating amount of a composition comprising aretro-inverso peptide having between 12 and about 40 amino acids,wherein the peptide includes the sequence that is retro-inverso withrespect to SEQ ID NO: 1, i.e. wherein said peptide comprises thesequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.Preferably, the peptide has the amino acid sequence that isretro-inverso with respect to SEQ ID NO: 1, i.e. wherein said peptidecomprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.Advantageously, the mammal is a human. In one aspect of this preferredembodiment, the administering step is direct local injection, systemic,intracranial, intracerebrospinal, topical or oral.

The present invention also provides a method for stimulatinghematopoiesis, comprising contacting pluripotential hematopoietic stemcells with an effective, hematopoiesis-stimulating amount of acomposition comprising a retro-inverso peptide having between 12 andabout 40 amino acids, wherein the peptide includes the sequence that isretro-inverso with respect to SEQ ID NO: 1, i.e. wherein said peptidecomprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.Preferably, the method results in the generation and differentiation ofmacrophages, neutrophils, eosinophils, basophils, mast cells,megakaryocytes or erythroid cells.

In another aspect of the present invention, there is provided aretro-inverso peptide having between 12 and about 40 amino acids,wherein said peptide includes the sequence that is retro-inverso withrespect to SEQ ID NO: 1, i.e. wherein said peptide comprises thesequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,for use in promoting neurite outgrowth or myelination in a mammal.Preferably, the peptide has the amino acid sequence shown in SEQ IDNO: 1. Advantageously, the mammal is a human.

The present invention also provides a retro-inverso peptide havingbetween 124-7 and about 40 amino acids, wherein the peptide includes thesequence that is retro-inverso with respect to SEQ ID NO: 1, i.e.wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,for use in stimulating hematopoiesis of pluripotential hematopoieticstem cells. Preferably, the peptide has the sequence that isretro-inverso with respect to SEQ ID NO: 1, i.e. wherein said peptidecomprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.

Another embodiment of the present invention is the use of aretro-inverso peptide having between 12 and about 40 amino acids,wherein the peptide includes the sequence that is retro-inverso withrespect to SEQ ID NO: 1, i.e. wherein said peptide comprises thesequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,in the preparation of a medicament for promoting neurite outgrowth ormyelination in a mammal in need thereof. Preferably, the peptide has thesequence that is retro-inverso with respect to SEQ ID NO: 1, i.e.wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.Advantageously, the mammal is a human.

The present invention also provides the use of a retro-inverso peptidehaving between 12 and about 40 amino acids, wherein the peptide includesthe sequence that is retro-inverso with respect to SEQ ID NO: 1, i.e.wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,in the preparation of a medicament for stimulating hematopoiesis ofpluripotential hematopoietic stem cells in a mammal in need thereof.Preferably, the peptide has the sequence that is retro-inverso withrespect to SEQ ID NO: 1, i.e. wherein said peptide comprises thesequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.Advantageously, the mammal is a human.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides retro-inverso (RI) peptides derived frominterleukin-3 (IL-3) which mediate similar effects to native IL-3,including stimulation of hematopoiesis and platelet production. The term“derived from” indicates that the peptides include an active region ofinterleukin 3, or analogs thereof as defined below. These RIIL-3-derived peptides stimulate the growth and differentiation ofmacrophages, neutrophils, eosinophils, basophils, mast cells,megakaryocytes and erythroid cells.

These peptides also have therapeutic applications in promotingfunctional recovery after toxic, traumatic, ischemic, degenerative andinherited lesions to the peripheral and central nervous system. Thesepeptides are also useful for promoting increased myelination and forcounteracting the effects of demyelinating diseases. The ability of aparticular retro-inverso peptide to mediate an effect similar to theparent peptide can be determined by a person of ordinary skill in theart using standard IL-3 assays as described in the examples below. Theuse of these peptides will facilitate treatment of various disorderssince they will be more stable and easier to synthesize than either thenative or recombinant cytokines.

A particular IL-3-derived peptide, from which the retro-inverso peptideof the invention is based is shown in Table 1. The corresponding native(non-retro-inverted) peptides is disclosed in U.S. Pat. No. 5,700,909,the entire contents of which are hereby incorporated by reference.

TABLE 1 Protein Name peptide sequence SEQ ID NO: humanIL-3 ILMENNLRRPNL1

As discussed above, these RI IL-3-derived peptides have the samehematopoietic activities as the corresponding full-length IL-3 protein,and also possess neurotrophic and myelinotrophic activity. Oneembodiment of the present invention is a method for stimulating thegeneration and differentiation of pluripotential hematopoietic stemcells into cells such as macrophages, neutrophils, eosinophils,basophils, mast cells, megakaryocytes and erythroid cells byadministering to the cells an effective, hematopoiesis-facilitatingamount of a RI peptide having between 12 and about 40 amino acids, andencompassing the peptide that is RI with respect to the IL-3-derivedpeptide shown in SEQ ID NO: 1, or analogs thereof which have similaractivity. Such analogs include, for example, replacement of one or morelysine and/or arginine residues with alanine or another amino acid;deletion of one or more lysine and/or arginine residues; replacement ofone or more tyrosine and/or phenylalanine residues, deletion of one ormore phenylalanine residues and conservative replacements of one or moreamino acids within the peptide. The replacement or deletion oflysine/arginine and tyrosine/phenylalanine residues will reduce thesusceptibility of peptide degradation by trypsin and chymotrypsin,respectively.

Additional variations of these peptide sequences contemplated for use inthe present invention include minor insertions and deletions.Conservative amino acid replacements are contemplated. Such replacementsare, for example, those that take place within a family of amino acidsthat are related in the chemical nature of their side chains. Thefamilies of amino acids include the basic charged amino acids (lysine,arginine, histidine); the acidic charged amino acids (aspartic acid,glutamic acid); the non-polar amino acids (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); theuncharged polar amino acids (glycine, asparagine, glutamine, cysteine,serine, threonine, tyrosine); and the aromatic amino acids(phenylalanine, tryptophan and tyrosine). In particular, it is generallyaccepted that conservative amino acid replacements consisting of anisolated replacement of a leucine with an isoleucine or valine, or anaspartic acid with a glutamic acid, or a threonine with a serine, or asimilar conservative replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the properties of thepeptide. The ability of any RI peptide comprising the sequence that isretro-inverso with respect to the sequence shown in SEQ ID NO: 1, orinsertions, deletions or substitutions thereof, to promote neuriteoutgrowth, myelination, reverse demyelination and prevent neural celldeath can be determined using the assays provided in the examplespresented below.

Various chemical modifications will improve the stability, bioactivityand ability of the peptide to cross the blood brain barrier. One suchmodification is aliphatic amino terminal modification with a derivativeof an aliphatic or aromatic acid, forming an amide bond. Suchderivatives include, for example, CH₃CO, CH₃(CH₂)_(n)CO (n=1-10),C₆H₅CH₂CO, H₂N—(CH₂)_(n)CO (n=1-10). Another modification is carboxyterminal modification with a derivative of an aliphatic or aromaticamine/alcohol coupled to the peptide via an amide/ester bond. Suchderivatives include those listed above. The peptides may also have bothamino and carboxy terminal modifications, wherein the derivatives areindependently selected from those listed above. The peptides may also beglycosylated, wherein either the alpha amino group or a D-Asn, or both,are modified with glucose or galactose. In another contemplatedmodification, selected backbone amide bonds are reduced (—NH—CH₂). Othermodifications include N-methylation of selected nitrogens in the amidebonds and esters in which at least one of the acid groups on the peptideare modified as aromatic or aliphatic esters. Any combination of theabove modifications is also contemplated.

Another embodiment of the present invention is a method of facilitatingneurite outgrowth in differentiated or undifferentiated neural cells bycontacting the cells with an effective, hematopoiesis-facilitatingamount of a RI peptide having between 12 and about 40 amino acids, andencompassing the sequence that is retro-inverso with respect to theIL-3-derived peptide shown in SEQ ID NO: 1, i.e. the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,or analogs thereof which have similar activity as described above.

The ability of any such peptide to stimulate neurite outgrowth caneasily be determined by one of ordinary skill in the art using theprocedures described in Examples 1-9 hereinbelow. The ability of anyparticular IL-3-derived peptide to mediate the same activity of nativeIL-3 can be determined using standard assays for the parent peptide asdiscussed in Examples 10-11.

A typical minimum amount of the RI peptides of the invention for theneurotrophic activity in cell growth medium is usually at least about 5ng/ml. This amount or more of the RI peptides of the invention for invitro use is contemplated. Typically, concentrations in the range of 0.1g/ml to about 10 g/ml of these peptides will be used. Effective amountsfor any particular tissue can be determined in accordance with Example1.

The hematopoietic or neural cells can be treated in vitro or ex vivo bydirectly administering the RI peptides of the invention to the cells.This can be done, for example, by culturing the cells in growth mediumsuitable for the particular cell type followed by addition of thepeptide to the medium. When the cells to be treated are in vivo,typically in a vertebrate, preferably a mammal, the composition can beadministered by one of several techniques. Most preferably, thecomposition is injected directly into the blood in sufficient quantityto give the desired local concentration of peptide. These RI peptidespersist longer in vivo due to the D peptide bonds. In the peptideslacking lysine and arginine residues, proteolytic degradation isreduced. The smaller peptides (i.e. 50-mer or less) will most likelycross the blood brain barrier and enter the central nervous system fortreatment of CNS disorders (see Banks et al., Peptides, 13: 1289-1294(1992)).

For treatment of neural disorders, direct intracranial injection orinjection into the cerebrospinal fluid may also be used in sufficientquantities to give the desired local concentration of neurotrophin. Inboth cases, a pharmaceutically acceptable injectable carrier is used.Such carriers include, for example, phosphate buffered saline andRinger's solution. Alternatively, the composition can be administered toperipheral neural tissue by direct local injection or by systemicadministration. Various conventional modes of administration arecontemplated including intravenous, pulmonary, intramuscular,intradermal, subcutaneous, intracranial, epidural, intrathecal, topicaland oral. Pharmaceutically acceptable carriers for topicaladministration include creams, gels, pastes, ointments, lotions,suspensions, emulsions and dispersions.

The peptide compositions of the invention can be packaged andadministered in unit dosage form such as an injectable composition orlocal preparation in a dosage amount equivalent to the daily dosageadministered to a patient or as a controlled release composition. Aseptum sealed vial containing a daily dose of the active ingredient ineither PBS or in lyophilized form is an example of a unit dosage. In apreferred embodiment, daily systemic dosages of the RI peptides of theinvention based on the body weight of the vertebrate for promoting IL-3effects such as stimulation of hematopoiesis, and for treatment ofneurodegenerative diseases or demyelination diseases, are in the rangeof from about 0.01 to about 10,000 g/kg. More preferably, daily systemicdosages are between about 0.1 and 1,000 g/kg. Most preferably, dailysystemic dosages are between about 10 and 100 g/kg. Daily dosages oflocally administered material will be about an order of magnitude less.Oral administration is particularly preferred because of the resistanceof the peptides to proteolytic degradation in the gastrointestinalsystem.

In one preferred embodiment of the invention, the peptides areadministered locally to neural cells in vivo by implantation thereof.For example, polylactic acid, polygalactic acid, regenerated collagen,multilamellar liposomes and many other conventional depot formulationscomprise bioerodible or biodegradable materials that can be formulatedwith biologically active neurotrophic peptide compositions. Thesematerials, when implanted, gradually break down and release the activematerial to the surrounding tissue. The use of bioerodible,biodegradable and other depot formulations is expressly contemplated inthe present invention. Infusion pumps, matrix entrapment systems andcombinations with transdermal delivery devices are also contemplated.The peptides may also be encapsulated within a polyethylene glycolconformal coating as described in U.S. Pat. No. 5,529,914 prior toimplantation.

The peptides of the invention may also be enclosed in micelles orliposomes. Liposome encapsulation technology is well known. Liposomesmay be targeted to specific tissue, such as neural tissue, through theuse of receptors, ligands or antibodies capable of binding the targetedtissue. The preparation of these formulations is well known in the art(Radin et al., Enzymol., 98: 613-618 (1983)).

There are currently no available pharmaceuticals able to promote fullfunctional regeneration and restoration of the structural integrity ofneural systems. This is particularly true of the CNS. Any degree ofregeneration of peripheral nerves through the use of neurotrophicfactors is within the scope of this invention. Moreover, neurotrophicfactors can be therapeutically useful in the treatment ofneurodegenerative diseases associated with the degeneration of neuralpopulations or specific areas of the brain. Any degree of retardation orhalting or reversing such degeneration is within the scope of thepresent invention. The principal cause of Parkinson's disease is thedegeneration of dopaminergic neurons of the substantia nigra. The RIpeptides of the invention may be therapeutically useful in the treatmentof Parkinson's disease. Retinal neuropathy, anocular neurodegenerativedisorder leading to loss of vision in the elderly, is also treatableusing the RI peptides of the invention.

It has long been believed that in order to reach neuronal populations inthe brain, neurotrophic factors would have to be administeredintracerebrally since these proteins do not cross the blood brainbarrier. U.S. Pat. No. 5,571,787 discloses that an iodinatedneurotrophic 18-mer fragment derived from saposin C crosses the bloodbrain barrier. Thus, the RI peptides having up to about 22 amino acidswill also cross this barrier and can thus be administered intravenously.Other neuronal populations, such as motor neurons, can also be treatedby intravenous injection, although direct injection into thecerebrospinal fluid is also envisioned as an alternate route.

Cells may be treated to facilitate myelin formation or to preventdemyelination in the manner described above in vivo, ex vivo or invitro. Diseases resulting in demyelination of nerve fibers including MS,acute disseminated leukoencephalitis, trauma to brain and/or spinalcord, progressive multifocal leukoencephalitis, metachromaticleukodystrophy, adrenal leukodystrophy and maldevelopment of the whitematter in premature infants (periventricular leucomalacia) can be slowedor halted by administration of the neurotrophic peptides of theinvention to the cells affected by the disease.

The RI IL-3-derived peptide compositions of the present invention canalso be used to support hematopoieses, to enhance the survival ofcultured motor neurons and to determine the effects of neurotrophicfactors and myelin facilitating materials. However, more practically,they have an immediate use as laboratory reagents and components of cellgrowth media in order to facilitate hematopoiesis and maintain neuralcells in vitro.

The peptides of the invention are synthesized using an automatedsolid-phase protocol well known in the art. All peptides are purified byhigh performance liquid chromatography (HPLC) on a reverse-phase columnto an extent greater than about 95% prior to use.

The following examples are merely illustrative and are not intended tolimit the scope of the present invention.

Example 1 Stimulation of Neurite Outgrowth

NS20Y neuroblastoma cells are grown in DMEM containing 10% fetal calfserum (FCS). Cells are removed with trypsin and plated in 30 mm petridishes onto glass coverslips. After 20-24 hours, the medium is replacedwith 2 ml DMEM containing 0.5% FCS plus 0, 0.5, 1, 2, 4 or 8 ng/ml of aRI IL-3-derived peptide having between 12 and about 40 amino acids andincluding the sequence that is retro-inverso with respect to SEQ IDNO: 1. Cells were cultured for an additional 24 hours, washed with PBSand fixed with Bouin's solution (saturated aqueous picricacid/formalin/acetic acid 15:5:1) for 30 minutes. Fixative was removedwith PBS and neurite outgrowth was scored under a phase contrastmicroscope. Cells exhibiting one or more clearly defined neurites equalto or longer than one cell diameter were scored as positive. At least200 cells were scored in different portions of each dish to determinethe percentage of neurite bearing cells and assays were performed induplicate.

Example 2 Prevention of Cell Death

NS20Y cells are plated as described in Example 1 and grown on glasscoverslips in 0.5% fetal bovine serum for 2 days in the presence orabsence of 8 ng/ml of an RI IL-3-derived peptide having between 12 andabout 40 amino acids and including the sequence that is retro-inversowith respect to SEQ ID NO: 1. Media is removed and 0.2% trypan blue inPBS is added to each well. Blue-staining dead cells are scored as apercentage of the total on an inverted microscope, counting 400 cells infour areas of each well. The average error of duplicates was 5%.

Example 3 Promotion of Neurite Outgrowth Ex Vivo

Dorsal root ganglia are removed from adult rats and sensory neurons wereprepared as described by Kuffler et al. (J. Neurobiol. 25:1267-1282,1994). Neurons are treated with 0.5 ng/ml of an RI IL-3-derived peptidehaving between 12 and about 40 amino acids and including the sequencethat is retro-inverso with respect to SEQ ID NO: 1. After three days oftreatment, the length of the longest neuritic projections are measuredon a micrometer grid. The longest neurites in neurons treated with RIpeptide are approximately three times longer than those treated with acontrol (non-RI) peptide or in untreated controls. After a 48 hourtreatment, all cells respond similarly to nerve growth factor (NGF) inthat extensive branching is observed. These results indicate that theIL-3-derived peptides promote the differentiation of sensory neurons.

Example 4 Reversal of Demyelination in a Rat Model

Experimental allergic encephalomyelitis (EAE) is a rat model of humanmultiple sclerosis (MS). In rats, EAE is induced by injecting foreignprotein (guinea pig spinal cord) which results in inflammation anddemyelination in white matter 11 days later. This demyelinationresembles that seen in actively demyelinating human MS lesions (Liu etal., Multiple Sclerosis, 1: 2-9 (1995)).

EAE is induced in Lewis rats by injection of an emulsion of guinea pigspinal cord and complete Freund's adjuvant (CFA). At day 14, whenweakness is evident, treatment with a RI IL-3-derived peptide havingbetween 12 and about 40 amino acids, and including the sequence that isretro-inverso with respect to SEQ ID NO: 1, is begun (200 g/kgintramuscularly) and continued for 8 days every day. Six rats areinjected with vehicle only. Stride length, a measure of muscle weakness,is scored on days 14 and 22. In addition, the number and size ofdemyelinating lesions (plaques) in the spinal cord at day 22 per mm² isscored. Lastly, the amount of cholesterol ester in brain, a marker ofmyelin breakdown, is scored at day 22.

The stride length of both groups is decreased at day 14, whereas aftertreatment for 8 days, the IL-3-derived peptide-treated animals return tonormal, but the vehicle treated animals do not. A significant reductionof cholesterol ester content is observed in the brains of the treatedgroup. Moreover, the number of spinal cord lesions is significantlyreduced after 10 days of treatment with IL-3-derived peptide. Lastly,the average lesion size is significantly reduced. There is no differencein weight loss between the control and experimental animals. Theseresults indicate a significant clinical, biochemical and morphologicalreversal of EAE after systemic treatment with IL-3-derived peptides.This action differs from the anti-inflammatory effect of current MSdrugs which do not act directly upon myelin repair.

Example 5 Ex Vivo Myelination Assay

Newborn mouse cerebellar explants are prepared according to Satomi(Zool. Sci. 9:127-137, 1992). Neurite outgrowth and myelination areobserved for 22 days in culture, during the period when the newbornmouse cerebellum normally undergoes neuronal differentiation andmyelination begins. An RI IL-3-derived peptide having between 12 andabout 40 amino acids, and including the sequence that is retro-inversowith respect to SEQ ID NO: 1, is added on the second day afterpreparation of the explants (three control and three treated explants)and outgrowth of neurites and myelination are assessed under a brightfield microscope with a video camera. Saposin C is used as a positivecontrol at a concentration of between about 1 and 10 g/ml. Myelinationis stimulated by the IL-3-derived peptides to a similar extent as withsaposin C.

Alternatively, myelination may be assayed by incorporation of ³⁵S intosulfolipids which are exclusive to myelin as described below.

Example 6 Incorporation of ³⁵S into Sulfolipids

Primary myelin-containing Schwann cells are incubated in low sulfatemedia (DMEM) containing 0.5% fetal bovine serum (FBS), followed byaddition of ³⁵S-methionine and an RI IL-3-derived peptide having between12 and about 40 amino acids, and including the sequence that isretro-inverso with respect to SEQ ID NO: 1, for 48 hours. Saposin C isused as a positive control. Cells are rinsed with PBS, harvested andsonicated in 100 l distilled water. An aliquot of cell lysate is removedfor protein analysis and the remainder is extracted with 5 mlchloroform/methanol (2:1, v/v). Lipid extracts are chromatographed andimmunostained with anti-sulfatide monoclonal antibody as described(Hiraiwa et al., Proc, Natl. Acad. Sci. U.S.A. 94: 4778-4781). Similaramounts of sulfatide are observed after peptide and saposin C treatment.

Example 7 Use of RI Peptides in Treating Traumatic Ischemic CNS Lesions

Humans with traumatic lesions to the brain or spinal cord receivesystemic injections of about 100 g/kg of an RI IL-3-derived peptidehaving between 12 and about 40 amino acids, and including the sequencethat is retro-inverso with respect to SEQ ID NO: 1, in a sterile salinesolution or in depot form. Improvement is assessed by gain of sensory ormotor nerve function (i.e. increased limb movement). Treatments continueuntil no further improvement occurs.

Example 8 Use of RI Peptides in Treating Demyelination Disorders

Patients diagnosed with early stage MS are given an RI IL-3-derivedpeptide having between 12 and about 40 amino acids, and including thesequence that is retro-inverso with respect to SEQ ID NO: 1, by systemicinjection using the same dose range as in Example 7. Dosages arerepeated daily or weekly and improvement in muscle strength,musculoskeletal coordination and myelination (as determined by MRI) isobserved. Patients with chronic relapsing MS are treated in the samemanner when subsequent relapses occur.

Typically, IL-3 is assayed by its ability to stimulate the formation ofcolonies of differentiated cells from progenitor cells of thebone-marrow in soft agar. Alternatively, IL-3 can be assayed for itsproliferative effect on cell lines derived from human leukemias such asTF-1 and MO-7e. Detailed protocols for IL-3 assays may be found inCytokines, a Practical Approach, Balkwill, F., ed., IRL Press, New York,second edition (1995) pp. 247-268, 372-377, the entire contents of whichare incorporated by reference. IL-3 assay protocols are provided in thefollowing example.

Example 9 IL-3 Colony Formation Assay

IL-3 stimulates the formation of mixed colonies of different cell types.Colonies of more than 50 cells are counted and analyzed after 7-14 daysby eye using a binocular microscope. The number of colonies is usuallyrelated to the specific activity or concentration of IL 3 in the agarculture. Morphological analysis by staining of dried and fixed gelsallows proper identification of the colony type (Metcalf, TheHemopoietic Colony Stimulating Factors, Elsevier Press, Amsterdam(1984)).

Cell suspensions are prepared from human bone marrow by collectingbone-marrow aspirates in sterile tubes containing 5-10 ml of Iscove'smodified Dulbecco's medium (IMDM) plus 400 units of preservative-freeheparin. Cells are centrifuged at 600-1,000×g for 10 min, thesupernatant is discarded and the cells are resuspended in IMDM plus 2%fetal calf serum (FCS), then mixed gently by pipetting. Cells arecounted in a hemacytometer and the concentration is adjusted as needed.As the cellularity of bone-marrow aspirates varies with the pathology ofthe patient, and also with the aspiration technique, some judgment mustbe exercised about the volume used to resuspend the cells. Hypocellularmarrow samples are resuspended in 1-2 ml, but usually 5-10 ml are used.Cell suspensions are kept on ice until plating.

The following protocol details the steps for the assay of Mix-CFC fromhuman bone marrow. This assay allows the growth of mixed colonies, whichmay contain several myeloid lineages (neutrophils, eosinophils,basophils, erythroid cells, monocytes-macrophages and megakaryocytes)resulting from clonal proliferation of Mix-CFC. Three aliquots of 1 mlcontaining 5×104 human bone marrow cells each are placed in 3 cmdiameter Petri dishes. The plating mixture is made as follows, to atotal value of 3.3 ml (to allow 0.3 ml for waste):

Component Vol (%) For 3.3 ml Cell suspension(10X desired 10 0.33 finalconcentration) BSA (10% stock solution) 10 0.33 IL-3 RI peptide or cond.medium 10 0.33 Fetal calf serum 20 0.66 Erytoropoietin (2 units) 20. 066IMDM 48 1.32

The agar (3.3%) is placed into a boiling water bath to melt. While theagar is melting, the plating mixture may be warmed to 37 C in a waterbath to prevent the agar from setting too quickly when added. Agar (0.30ml) is added, mixed thoroughly but gently, and 1 ml is plated per dish.Dishes are placed on a tray and allowed to set. The dishes can be placedin a refrigeration for about 2 min. to speed the process. The plates arethen placed into a fully humidified gas incubator at 37 C and incubatedfor 14 days. Colonies are scored under about 40× magnification using amicroscope with a zoom lens, or an inverted microscope. Individualcolonies are picked up for cytological examination using a Pasteurpipette and suspended in 0.1 ml of medium (plus a source of protein,either 1% serum or 0.1% BSA, for standard cytospin preparations.

Example 10 IL-3 Cell Proliferation Assay

Several IL-3-dependent cell lines can be employed to determine whether apeptide has IL-3 activity. The cell lines FDCP-1, FDCP-2, 32DCL23, TF-1(human erythroleukemia), AML-193 (human acute myeloid leukemia; ATCC CRL9589); MO-7e (human megakaryoblastic leukemia) and DAM1 (humanmegakaryoblastic cells; Chen et al., Br. J. Haematol. 88: 481-490, 1994)are all IL-3-dependent. To determine whether a particular RIIL-3-derived peptide having between 12 and about 40 amino acids, andincluding the sequence that is retro-inverso with respect to SEQ ID NO:1, has IL-3 activity, the peptide or analog thereof such as a peptidehaving one or more conservative amino acid replacements, is assayed forIL-3 activity by incubation with cells as follows:

Cells are cultured in suspension until they reach an exponential growthrate, then harvested by spinning at 800×g for 5 minutes on a benchcentrifuge using a swing-out rotor. The cell pellet is suspended in theappropriate medium, centrifuged again, and resuspended a further twotimes. Cells are resuspended at a concentration of 1-2×10⁶/mol inFischer's medium (Gibco-plus glutamine) plus horse serum (10% v/v) andplated out in a total volume of 100 l at trial concentrations of 1-2×10⁵cells/ml with either dilutions of the RI IL-3-derived peptide under testor dilutions of a standard preparation of IL-3. The final concentrationof horse serum (or, if the cells are normally cultured in it, fetal calfserum) is 10-20% (v/v). Cells are incubated in a CO₂ incubator at 37 C.

After 24 or 48 hours, the effects of the growth factors on survival andproliferation are assessed using the Trypan blue exclusion assay inwhich the cell suspension is mixed 1:1 with Trypan blue solution and theviable cells which exclude the dye are counted. Another method forassessment of cell proliferation (DNA synthesis) involves themeasurement of [3H] thymidine incorporation. After 24 or 48 hours, [³H]thymidine (37 kBq) is added to each well and the incubation continuedfor 4 hours. The cells are removed from the incubator and seriallytransferred to GF/C filters on a Millipore cell harvester or similarapparatus. The cells are washed three times with trichloroacetic acid(TCA) and the TCA precipitable material retained on the filter countedin a liquid scintillation counter. The relative growth promotingactivities of the standard and the diluents of the RI IL-3-derivedpeptides are compared to quantify the growth promoting activity of theRI IL-3-derived peptides.

It should be noted that the present invention is not limited to onlythose embodiments described in the Detailed Description. Any embodimentwhich retains the spirit of the present invention should be consideredto be within its scope. However, the invention is only limited by thescope of the following claims.

1. A method for promoting neurite outgrowth or myelination in a mammalin need thereof comprising the step of administering to said mammal aneffective, neurite outgrowth or myelination-facilitating amount of acomposition comprising isolated retro-inverso peptide having between 12and about 40 amino acids, wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ileand a pharmaceutically acceptable carrier.
 2. The method of claim 1,wherein said retro-inverso peptide consists of the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.3. The method of claim 1 or 2, wherein said mammal is a human.
 4. Themethod of claim 1 or 2, wherein said administering step is selected fromthe group consisting of direct local injection, systemic, intracranial,intracerebrospinal, topical and oral.
 5. A method for stimulatinghematopoiesis comprising contacting pluripotential hematopoietic stemcells with an effective, hematopoiesis-stimulating amount of acomposition comprising isolated retro-inverso peptide having between 12and about 40 amino acids, wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ileand a pharmaceutically acceptable carrier.
 6. The method of claim 5,wherein said retro-inverso peptide consists of the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.7. The method of claim 5 or 6, wherein said method results in thegeneration and differentiation of cells selected from the groupconsisting of macrophages, neutrophils, eosinophils, basophils, mastcells, megakaryocytes and erythroid cells.
 8. A method for promotingneurite outgrowth or myelination in a mammal in need thereof comprisingthe step of administering to said mammal an effective, neurite outgrowthor myelination-facilitating amount of a composition comprising isolatedretro-inverso peptide having between 12 and about 40 amino acids,wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,wherein said peptide is optionally modified, said modification selectedfrom a group consisting of glycosylation, reduction of one or more amidebonds, methylation of one or more nitrogens, esterification of one ormore carboxylic acid groups, and modification at the amino terminus,carboxy terminus, or both amino and carboxy termini with a moietyindependently selected from the group consisting of CH₃CO,CH₃(CH₂)_(n)CO, C₆H₅CH₂CO and H₂N(CH₂)_(n)CO, wherein n=1-10, and apharmaceutically acceptable carrier.
 9. The method of claim 8, whereinsaid retro-inverso peptide consists of the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.10. The method of claim 8 or 9, wherein said mammal is a human.
 11. Themethod of claim 10, wherein said administering step is selected from thegroup consisting of direct local injection, systemic, intracranial,intracerebrospinal, topical and oral.
 12. A method for stimulatinghematopoiesis comprising contacting pluripotential hematopoietic stemcells with an effective, hematopoiesis-stimulating amount of acomposition comprising isolated retro-inverso peptide having between 12and about 40 amino acids, wherein said peptide comprises the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile,wherein said peptide is optionally modified, said modification selectedfrom a group consisting of glycosylation, reduction of one or more amidebonds, methylation of one or more nitrogens, esterification of one ormore carboxylic acid groups, and modification at the amino terminus,carboxy terminus, or both amino and carboxy termini with a moietyindependently selected from the group consisting of CH₃CO,CH₃(CH₂)_(n)CO, C₆H₅CH₂CO and H₂N(CH₂)_(n)CO, wherein n=1-10, and apharmaceutically acceptable carrier.
 13. The method of claim 12, whereinsaid retro-inverso peptide consists of the sequenceD-Leu-D-Asn-D-Pro-D-Arg-D-Arg-D-Leu-D-Asn-D-Asn-D-Glu-D-Met-D-Leu-D-Ile.14. The method of claim 12 or 13, wherein said method results in thegeneration and differentiation of cells selected from the groupconsisting of macrophages, neutrophils, eosinophils, basophils, mastcells, megakaryocytes and erythroid cells.